GSM6934154: PA3; Homo sapiens; RNA-Seq
Fresh tumor material from pLGG biopsies was minced with scalpels on ice and then subjected to enzymatic dissociation, using a freshly prepared papain solution (PAP2 Papain Dissociation System; CellSystems) in DMEM/F12 medium supplemented with 1000 U DNase I from bovine pancreas (Roche). After incubation at 37°C in 5% CO2, the solution was filtered using a 40 µm cell strainer, and red blood cells were lysed with ACK lysis buffer (Thermo Fisher Scientific) for 5 minutes on ice. The resulting single cell suspension was stained with 7-AAD Viability Staining Solution (Thermo Fisher Scientific), and non-viable, 7-AAD-positive cells were removed by fluorescence-activated cell sorting using a FACSAria II cell sorter (BD Biosiences). Approximately 10,000 vital cells of each sample were processed for scRNA-seq using using 10x Genomics Chromium Next GEM Single Cell 3' Technology (v3.1 Chemistry; 10x Genomics) according to the manufacturer's instructions. Quality, purity, average size and concentration of copy DNAs and libraries were determined by measurements on a Tapestation 2200 (Agilent Technologies). Indexed libraries (Single Index Kit T) were sequenced using a NextSeq 2000 sequencing platform with P3 flow cell and reagents (Illumina).